Project Abstract The Sonic Hedgehog (SHH) signaling pathway plays critical roles in the genesis of a large number of human cancers, including medulloblastoma. To date, the majority of small molecule inhibitors that block SHH signaling have targeted the pivotal upstream activator Smoothened (SMO), which regulates the levels and activity of the GLI family of transcription factors. Two of these compounds, including vismodegib, are FDA-approved for metastatic basal cell carcinoma patients, and are now undergoing clinical evaluation in MB patients. However, inherent resistance due to mutations downstream of SMO, or rapid tumor recurrence, has already been frequently observed in MB patients treated with vismodegib. Further, genomic analysis of relapsed, vismodegib resistant basal cell carcinomas has revealed similar mechanisms of cross-resistance to structurally diverse SMO inhibitors. Such data highlight the urgent need for targeted therapies with distinct mechanisms of action, with which to treat patients harboring SHH-driven cancers and to prevent their tumor recurrence. The goal of this proposal is to identify and characterize a novel set of such regulators (Aims 1 & 2), and to validate their in vivo roles using mouse and human orthotopic mouse models of MB (Aim 3). The work proposed herein will address the substantial clinical need for novel druggable targets in SHH-driven cancers. The combined experience of our team, our extensive unpublished findings, and the unique resources we bring to this project will prove pivotal in successful completion of the proposed Aims.